August 30, 2016 SPADA Meeting Book
Pre-decisional Draft, Do Not Distribute
Annex IV: Bioinformatics Analyses of Signature Sequences underlying Variola Virus Assays 117 118 In silico screening will be performed on signature sequences (eg: oligo primers) to demonstrate 119 specificity to Variola virus and inclusivity across all sequenced Variola virus strains. 120 121 In silico results are suggestive of potential performance issues, so will guide necessary additions to 122 the wet screening panels. In silico identification of potential cross-reactions (false positives) or non- 123 verifications (false negatives) would require the affected strains be included in the exclusivity or 124 inclusivity panels, respectively, if available. 125 126 A vendor-selected tool to carry out the bioinformatics evaluation should be able to predict 127 hybridization events between signature components and a sequence in a database including 128 available genomic sequence data, using public genbank nt [http://www.ncbi.nlm.nih.gov/genbank/]. 129 The selected tool should be able to identify predicted hybridization events based on platform 130 annealing temperatures, thus ensuring an accurate degree of allowed mismatch is incorporated in 131 predictions. The program should detect possible amplicons from any selected database of sequence. 132 133 Potential tools for in silico screening of real-time PCR signatures include:
134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
• http://sourceforge.net/projects/simulatepcr/files/?source=navbar
o This program will find all possible amplicons and real time fluorescing events
from any selected database of sequence.
• NCBI tools
The vendor submission should include:
• Description of sequence databases used in the in silico analysis
• Description of conditions used for in silico analysis
o Stringency of in silico analysis must match bench hybridization conditions
• Description of tool used for bioinformatics evaluation
o Data demonstrating the selected tool successfully predicts specificity that has been
confirmed by wet-lab testing on designated isolates
These data can be generated retrospectively using published assays
• List of additional strains to be added to the inclusivity (Annex II) or exclusivity (Annex III)
panels based on the bioinformatics evaluation
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7 Draft SMPR for Variola for DoD
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